Tumor necrosis factor related ligand

ABSTRACT

Tumor Necrosis Factor Related Ligand (TRELL) polypeptides, a novel member of the tumor necrosis factor family (TNF) and compositions comprising them are disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/978,203, filed Oct. 29, 2004 (now U.S. Pat. No. 7,109,298), which isa divisional of U.S. patent application Ser. No. 09/245,198, filed Feb.5, 1999 (now U.S. Pat. No. 7,129,061), which is a continuation ofInternational Application number PCT/US97/13945, filed Aug. 7, 1997,which claims benefit of U.S. provisional application Nos. 60/023,541,filed Aug. 7, 1996; 60/028,515, filed Oct. 18, 1996; and 60/040,820,filed Mar. 18, 1997, the disclosures of which are incorporated byreference herein in their entireties.

TECHNICAL FIELD

The present invention relates to Tumor Necrosis Factor Related ligand or“TRELL,” a polypeptide which is a member of the Tumor Necrosis FactorFamily. The protein or its receptor may have anti-cancer and/orimmunoregulatory applications. Furthermore, cells transfected with thegene for TRELL may be used in gene therapy to treat tumors, autoimmuneand inflammatory diseases or inherited genetic disorders.

The invention described herein was made in part during the course ofwork under the grant #31-42275.94 and 32-41729.94 to Irene Garcia fromthe Swiss National Fund. Reserved rights described in paragraphs #28 and#29 of the Swiss National Fund statute.

BACKGROUND

The tumor-necrosis factor (TNF)-related cytokines are mediators of hostdefense and immune regulation. Members of this family exist inmembrane-anchored forms, acting locally through cell-to-cell contact, oras secreted proteins capable of diffusing to more distant targets. Aparallel family of receptors signals the presence of these moleculesleading to the initiation of cell death or cellular proliferation anddifferentiation in the target tissue. Presently, the TNF family ofligands and receptors has at least nine recognized receptor-ligandpairs, including: TNF:TNF-R; LT-α:TNF-R; LT-α/β:LT-β-R; FasL:Fas;CD40L:CD40; CD30L:CD30; CD27L:CD27; OX40L:OX40 and 4-1BBL:4-1BB. The DNAsequences encoding these ligands have only about 25% to about 30%identity in even the most related cases, although the amino acidrelatedness is about 50%.

The defining feature of this family of cytokine receptors is found inthe cysteine rich extracellular domain initially revealed by themolecular cloning of two distinct TNF receptors.^(i) This family ofgenes encodes glycoproteins characteristic of Type I transmembraneproteins with an extracellular ligand binding domain, a single membranespanning region and a cytoplasmic region involved in activating cellularfunctions. The cysteine-rich ligand binding region exhibits a tightlyknit disulfide linked core domain, which, depending upon the particularfamily member, is repeated multiple times. Most receptors have fourdomains, although there may be as few as three, or as many as six.

Proteins in the TNF family of ligands are characterized by a shortN-terminal stretch of normally short hydrophilic amino acids, oftencontaining several lysine or arginine residues thought to serve as stoptransfer sequences. Next follows a transmembrane region and anextracellular region of variable length, that separates the C-terminalreceptor binding domain from the membrane. This region is sometimesreferred to as the “stalk.” The C-terminal binding region comprises thebulk of the protein, and often, but not always, contains glycosylationsites. These genes lack the classic signal sequences characteristic oftype I membrane proteins, having type II membrane proteins with the Cterminus lying outside the cell, and the short N-terminus residing inthe cytoplasm. In some cases, e.g., TNF and LT-α, cleavage in the stalkregion can occur early during protein processing and the ligand is thenfound primarily in secreted form. Most ligands, however, exist in amembrane form, mediating localized signalling.

The structure of these ligands has been well-defined by crystallographicanalyses of TNF, LT-α, and CD40L. TNF and lymphotoxin-α (LT-α) are bothstructured into a sandwich of two anti-parallel β-pleated sheets withthe “jelly roll” or Greek key topology.^(ii) The rms deviation betweenthe Cα and β-strand residues is 0.61 C, suggesting a high degree ofsimilarity in their molecular topography. A structural feature emergingfrom molecular studies of CD40L, TNF and LT-α is the propensity toassemble into oligomeric complexes. Intrinsic to the oligomericstructure is the formation of the receptor binding site at the junctionbetween the neighboring subunits creating a multivalent ligand. Thequaternary structures of TNF, CD40L and LT-α have been shown to exist astrimers by analysis of their crystal structures. Many of the amino acidsconserved between the different ligands are in stretches of the scaffoldβ-sheet. It is likely that the basic sandwich structure is preserved inall of these molecules, since portions of these scaffold sequences areconserved across the various family members. The quaternary structuremay also be maintained since the subunit conformation is likely toremain similar.

TNF family members can best be described as master switches in theimmune system controlling both cell survival and differentiation. OnlyTNF and LTα are currently recognized as secreted cytokines contrastingwith the other predominantly membrane anchored members of the TNFfamily. While a membrane form of TNF has been well-characterized and islikely to have unique biological roles, secreted TNF functions as ageneral alarm signaling to cells more distant from the site of thetriggering event. Thus TNF secretion can amplify an event leading to thewell-described changes in the vasculature lining and the inflammatorystate of cells. In contrast, the membrane bound members of the familysend signals though the TNF type receptors only to cells in directcontact For example T cells provide CD40 mediated “help” only to those Bcells brought into direct contact via cognate TCR interactions. Similarcell-cell contact limitations on the ability to induce cell death applyto the well-studied Fas system.

The ability to induce programmed cell death is an important andwell-studied feature of several members of the TNF family. Fas mediatedapoptosis appears to play a role in the regulation of autoreactivelymphocytes in the periphery and possibly the thymus (Castro et al.,1996) and recent work has also implicated the TNF and CD30 systems inthe survival of T cells and large cell anaplastic lymphoma lines(Amakawa et al., 1996; Gruss et al., 1994; Sytwu et al., 1996; Zheng etal., 1995). We and others had previously shown the death of this line inresponse to TNF, Fas or LTh receptor signaling to have features ofapoptosis (Abreu-Martin et al., 1995; Browning et al., 1996).

It appears that one can segregate the TNF ligands into three groupsbased on their ability to induce cell death (Table III). First, TNF, Fasligand and TRAIL can efficiently induce cell death in many lines andtheir receptors mostly likely have good canonical death domains.Presumably the ligand to DR-3 (TRAMP/WSL-1) would also all into thiscategory. Next there are those ligands which trigger a weaker deathsignal limited to few cell types and TRELL, CD30 ligand and LTa1b2 areexamples of this class. How this group can trigger cell death in theabsence of a canonical death domain is an interesting question andsuggests that a separate weaker death signaling mechanism exists.Lastly, there those members that cannot efficiently deliver a deathsignal. Probably all groups can have antiproliferative effects on somecell types consequent to inducing cell differentiation e.g. CD40(Funakoshi et al., 1994)

The TNF family has grown dramatically in recent years to encompass atleast 11 different signaling pathways involving regulation of the immunesystem. The expression patterns of TRELL and TRAIL indicate that thereis still more functional variety to be uncovered in this family. Thisaspect has been especially highlighted in recent the discovery of tworeceptors that affect the ability of rous sacroma and herpes simplexvirus to replicate as well as the historical observations that TNF hasanti-viral activity and pox viruses encode for decoy TNF receptors(Brojatsch et al., 1996; Montgomery et al., 1996; Smith, 1994; Vassalli,1992). The generation soluble TRELL and the identification of the TRELLreceptor should provide the tools to elucidate the biological functionof this interesting protein.

TNF is a mediator of septic shock and cachexia^(iii), and is involved inthe regulation of hematopoietic cell development.^(iv) It appears toplay a major role as a mediator of inflammation and defense againstbacterial, viral and parasitic infections^(v) as well as havingantitumor activity.^(vi) TNF is also involved in different autoimmunediseases.^(vii) TNF may be produced by several types of cells, includingmacrophages, fibroblasts, T cells and natural killer cells.^(viii) TNFbinds to two different receptors, each acting through specificintracellular signaling molecules, thus resulting in different effectsof TNF.^(ix) TNF can exist either as a membrane bound form or as asoluble secreted cytokine.^(x)

LT-α shares many activities with TNF, i.e. binding to the TNFreceptors,^(xi) but unlike TNF, appears to be secreted primarily byactivated T cells and some β-lymphoblastoid tumors.^(xii) Theheteromeric complex of LT-α and LT-β is a membrane bound complex whichbinds to the LT-β receptor.^(xiii) The LT system (LTs and LT-R) appearsto be involved in the development of peripheral lymphoid organs sincegenetic disruption of LT-β leads to disorganization of T and B cells inthe spleen and an absence of lymph nodes.^(xiv) The LT-β system is alsoinvolved in cell death of some adenocarcinoma cell lines.^(xv)

Fas-L, another member of the TNF family, is expressed predominantly onactivated T cells.^(xvi) It induces the death of cells bearing itsreceptor, including tumor cells and HTV-infected cells, by a mechanismknown as programmed cell death or apoptosis.^(xvii) Furthermore,deficiencies in either Fas or Fas-L may lead to lymphoproliferativedisorders, confirming the role of the Fas system in the regulation ofimmune responses. The Fas system is also involved in liver damageresulting from hepatitis chronic infection^(xix) and in autoimmunity inHIV-infected patients.^(xx) The Fas system is also involved in T-celldestruction in HIV patients.^(xxi) TRAIL, another member of this family,also seems to be involved in the death of a wide variety of transformedcell lines of diverse origin.^(xxii)

CD40-L, another member of the TNF family, is expressed on T cells andinduces the regulation of CD40-bearing B cells.^(xiii) Furthermore,alterations in the CD40-L gene result in a disease known as X-linkedhyper-IgM syndrome.^(xxiv) The CD40 system is also involved in differentautoimmune diseases^(xxv) and CD40-L is known to have antiviralproperties.^(xxvi) Although the CD40 system is involved in the rescue ofapoptotic B cells,^(xxvii) in non-immune cells it inducesapoptosis.^(xxviii) Many additional lymphocyte members of the TNF familyare also involved in costimulation.^(xxix)

Generally, the members of the TNF family have fundamental regulatoryroles in controlling the immune system and activating acute host defensesystems. Given the current progress in manipulating members of the TNFfamily for therapeutic benefit, it is likely that members of this familymay provide unique means to control disease. Some of the ligands of thisfamily can directly induce the apoptotic death of many transformed cellseg. LT, TNF, Fas ligand and TRAIL (Nagata, 1997). Fas and possibly TNFand CD30 receptor activation can induce cell death in nontransformedlymphocytes which may play an immunoregulatory function (Amakawa et al.,1996; Nagata, 1997; Sytwu et al., 1996; Zheng et al., 1995). In general,death is triggered following the aggregation of death domains whichreside on the cytoplasmic side of the TNF receptors. The death domainorchestrates the assembly of various signal transduction componentswhich result in the activation of the caspase cascade (Nagata, 1997).Some receptors lack canonical death domains, e.g. LTb receptor and CD30(Browning et al., 1996; Lee et al., 1996) yet can induce cell death,albeit more weakly. It is likely that these receptors function primarilyto induce cell differentiation and the death is an aberrant consequencein some transformed cell lines, although this picture is unclear asstudies on the CD30 null mouse suggest a death role in negativeselection in the thymus (Amakawa et al., 1996). Conversely, signalingthrough other pathways such as CD40 is required to maintain cellsurvival. Thus, there is a need to identify and characterize additionalmolecules which are members of the TNF family thereby providingadditional means of controlling disease and manipulating the immunesystem.

SUMMARY

Accordingly, the present invention is directed to a polypeptide, a tumornecrosis factor related ligand called TRELL which substantially obviatesone or more of the problems due to the limitations and disadvantages ofthe related art. The inventor has discovered a new member of the TNFfamily of cytokines, and defined both the human and murine amino acidsequence of the protein, as well as the DNA sequences encoding theseprotein. The claimed invention may be used to identify new diagnosticsand therapeutics for numerous diseases and conditions as discussed inmore detail below, as well as to obtain information about, andmanipulate, the immune system and its processes. Additionally, theclaimed invention is involved in the induction of cell death incarcinoma.

Additional features and advantages of the invention will be set forth inthe description which follows, and in part will be apparent form thedescription, or may be learned by practice of the invention. Theobjectives and other advantages of the invention will be realized andattained by the compositions and methods particularly pointed out in thewritten description and claims hereof, as well as in the appendeddrawings.

Thus, to achieve these and other advantages, and in accordance with thepurpose of the invention, as embodied and broadly described herein, theinvention includes a DNA sequence encoding TRELL. The nucleotidesequence for mouse TRELL (mTRELL) is shown in SEQ ID. NO. 1, and forhuman TRELL (hTRELL) in SEQ ID. NO. 3. Specifically, the inventionrelates to DNA sequences which encode a TRELL having the amino acidsequence identified in SEQ. ID. NO. 2 (mTRELL) or 4 (hTRELL). In otherembodiments, the invention relates to sequences that have at least 50%homology with the DNA encoding the C terminal receptor binding domain ofTRELL and hybridize to the claimed DNA sequences or fragments thereof,and which encode TRELL having the sequence identified in SEQ. ID. NO. 4or SEQ ID. NO. 2.

The invention in certain embodiments furthermore relates to a DNAsequence encoding TRELL where the sequence is operatively linked to anexpression control sequence. Any suitable expression control sequence isuseful in the claimed invention, and can easily be selected by oneskilled in the art.

The invention also contemplates a recombinant DNA comprising a sequenceencoding TRELL, or a fragment thereof, as well as hosts with stablyintegrated TRELL sequences introduced into their genome, or possessingepisomal elements. Any suitable host may be used in the invention, andcan easily be selected by one skilled in the art without undueexperimentation.

In other embodiments, the invention relates to methods of producingsubstantially pure TRELL comprising the step of culturing transformedhosts, and TRELL essentially free of normally associated animalproteins.

The invention encompasses TRELL having the amino acid sequenceidentified in SEQ. ID. NO. 4 or SEQ ID. NO. 2 as well as fragments orhomologs thereof. In various embodiments, the amino acid and/or the DNAsequence of TRELL may comprise conservative insertions, deletions andsubstitutions, as further defined below or may comprise fragments ofsaid sequences.

The invention relays in other embodiments to soluble TRELL constructs,which may be used to directly trigger TRELL mediated pharmacologicalevents. Such events may have useful therapeutic benefit in the treatmentof cancer or the manipulation of the immune system to treat immunologicdiseases. Soluble TRELL forms could be genetically reengineered toincorporate an easily recognizable tag, thereby facilitating theidentification of TRELL receptors.

In yet other embodiments the invention relates to methods of genetherapy using the TRELL's disclosed and claimed herein.

The pharmaceutical preparations of the invention may, optionally,include pharmaceutically acceptable carriers, adjuvants, fillers, orother pharmaceutical compositions, and may be administered in any of thenumerous forms or routes known in the art.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory, andare intended to provide further explanation of the invention as claimed.

The accompanying drawings are included to provide a furtherunderstanding of the invention, and are incorporated in, and constitutea part of this specification, illustrate several embodiments of theinvention, and together with the description serve to explain theprinciples of the invention.

DESCRIPTION OF DRAWINGS

FIG. 1 is an amino acid sequence comparison of human (SEQ ID NO: 4) andmouse (SEQ ID NO: 2) TRELL.

FIGS. 2A and 2B are an amino acid comparison of human members of the TNFfamily including: hTNF (SEQ ID NO: 19); hLT-alpha (SEQ ID NO: 20);hLT-beta (SEQ ID NO: 21); hFasL (SEQ ID NO: 22); hTFRP (SEQ ID NO: 4);hTRAIL (SEQ ID NO: 23); hcD27L (SEQ ID NO: 24); hCD30L (SEQ ID NO: 25);hCD40L (SEQ ID NO: 26); h4-1BBL (SEQ ID NO: 27).

FIG. 3 is a northern analysis of TRELL mRNA expression in differentmouse cell lines and tissues. Lanes are duplicated and contained RNAfrom (1) thioglycolate induced peritoneal macrophages, (2) bone marrow,(3) spleen, and (4) liver.

FIG. 4 is a northern analysis of TRELL mRNA expression in differenthuman tissues.

FIG. 5: SDS-PAGE of recombinant TNF, LTα and TRELL (designated here as“TWEAK”) under reducing and nonreducing conditions.

FIG. 6: TRELL is cytotoxic to the human adenocarcinoma line HT29.

A. Ability of the TNF, TRELL, LTα/β and anti-Fas to block the growth ofthe HT29 line in the presence of human interferon-γ. Cells were grownfor 4 days in the presence of the various agents and growth was assessedusing MTT staining.

B. Morphology of the cells undergoing cell death. Cells were pregrownfor 2 days and then treated for 24 hours with 80 U/mL interferon-g withno further addition (A) or the addition of 100 ng/mL recombinant TRELL(B). Cells were fixed with ethanol and stained with 1 μg/mL Hoescht dyeto reveal the nuclei. Top panels show phase contrast images and thebottom panels show Hoescht dye stained chromatin.

DETAILED DESCRIPTION

Reference will now be made in detail to the present preferredembodiments of the invention. This invention relates to DNA sequencesthat code for human or mouse TRELL, fragments and homologs thereof, andexpression of those DNA sequences in hosts transformed with them. Theinvention relates to uses of these DNA sequences and the peptidesencoded by them. Additionally, the invention encompasses both human andmouse amino acid sequences for TRELL, or fragments thereof, as well aspharmaceutical compositions comprising or derived from them.

A. DEFINITIONS

“Homologous”, as used herein, refers to the sequence similarity betweensequences of molecules being compared. When a position in both of thetwo compared sequences is occupied by the same base or amino acidmonomer subunit, e.g., if a position in each of two DNA molecules isoccupied by adenine, then the molecules are homologous at that position.The percent of homology between two sequences is a function of thenumber of matching or homologous positions shared by the two sequencesdivided by the number of positions compared×100. For example, if 6 of10, of the positions in two sequences are matched or homologous then thetwo sequences are 60% homologous. By way of example, the DNA sequencesATTGCC and TATGGC share 50% homology. Generally, a comparison is madewhen two sequences are aligned to give maximum homology.

A “purified preparation” or a “substantially pure preparation” of apolypeptide, as used herein, means a polypeptide that has been separatedfrom other proteins, lipids, and nucleic acids with which it naturallyoccurs. Preferably, the polypeptide is also separated from othersubstances, e.g., antibodies, matrices, etc., which are used to purifyit.

“Transformed host” as used herein is meant to encompass any host withstably integrated sequence, i.e. TRELL sequence, introduced into itsgenome or a host possessing sequence, i.e. TRELL, encoding episomalelements.

A “treatment”, as used herein, includes any therapeutic treatment, e.g.,the administration of a therapeutic agent or substance, e.g., a drug.

A “substantially pure nucleic acid”, e.g., a substantially pure DNA, isa nucleic acid which is one or both of: not immediately contiguous witheither one or both of the sequences, e.g., coding sequences, with whichit is immediately contiguous (i.e., one at the 5′ end and one at the 3′end) in the naturally-occurring genome of the organism from which thenucleic acid is derived; or (2) which is substantially free of a nucleicacid sequence with which it occurs in the organism from which thenucleic acid is derived. The term includes, for example, a recombinantDNA which is incorporated into a vector, e.g., into an autonomouslyreplicating plasmid or virus, or into the genomic DNA of a prokaryote oreukaryote, or which exists as a separate molecule (e.g., a cDNA or agenomic DNA fragment produced by PCR or restriction endonucleasetreatment) independent of other DNA sequences. Substantially pure DNAalso includes a recombinant DNA which is part of a hybrid gene encodingTRELL.

The terms “peptides”, “proteins”, and “polypeptides” are usedinterchangeably herein.

“Biologically active” as used herein, means having an in vivo or invitro activity which may be performed directly or indirectly.Biologically active fragments of TRELL may have, for example, 70% aminoacid homology with the active site of TRELL, more preferably at least80%, and most preferably, at least 90% homology. Identity or homologywith respect to TRELL is defined herein as the percentage of amino acidresidues in the candidate sequence which are identical to the TRELLresidues in SEQ. ID. NOS. 2 or 4.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art. Such techniquesare described in the literature.^(xxx)

B. DNA SEQUENCES OF THE INVENTION

As described herein, one aspect of the invention features asubstantially pure (or recombinant) nucleic acid which includes anucleotide sequence encoding a TRELL polypeptide, such as the DNAdescribed in SEQ. ID. NO. 1 or 3 and/or equivalents of such nucleicacids. The term nucleic acid as used herein can include fragments andequivalents, such as, for example, sequences encoding functionallyequivalent peptides. Equivalent nucleotide sequences may includesequences that differ by one or more nucleotide substitutions, additionsor deletions, such as allelic variants, mutations, etc. and includesequences that differ from the nucleotide sequence encoding TRELL shownin SEQ ID NO: 1 or 3, due to the degeneracy of the genetic code. Theinventors have sequenced a human 1936 bp DNA which contains an openreading frame encoding a TRELL polypeptide, having the 248 amino acidsequence as identified in SEQ. ID. NO. 4.

The inventor describes herein both human and murine sequences; theinvention will be described generally by reference to the humansequences, although one skilled in the art will understand that themouse sequences are encompassed herein. A striking feature of TRELL isthe extensive sequence conservation of the receptor binding domainbetween mouse and man; only the Fas ligand approaches this level ofconservation. Both the murine and human TRELL proteins have all of thecharacteristics of the TNF family, i.e., a type II membrane proteinorganization and conservation of the sequence motifs involved in thefolding of the protein into the TNF ant-parallel β-sheet structure.

The nucleotide sequence for mTRELL is set forth in SEQ. ID. NO. 1; theamino acid sequence for mTRELL is described in SEQ. ID. NO. 2. The DNAand amino acid sequences for hTRELL are described in SEQ. ID. NOS. 3 and4 respectively.

The sequences of the invention can be used to prepare a series of DNAprobes that are useful in screening various collections of natural andsynthetic DNAs for the presence of DNA sequences that code for TRELL orfragments or derivatives thereof. One skilled in the art will recognizethat reference to “TRELL”, as used herein, refers also to biologicallyactive derivatives, fragments or homologs thereof.

The DNA sequences encoding TRELL of the invention can be employed toproduce TRELL peptides on expression in various prokaryotic andeukaryotic hosts transformed with them. These TRELL peptides may be usedin anti-cancer, and immunoregulatory applications. In general, thiscomprises the steps of culturing a host transformed with a DNA moleculecontaining the sequence encoding TRELL, operatively-linked to anexpression control sequence.

The DNA sequences and recombinant DNA molecules of the present inventioncan be expressed using a wide variety of host/vector combinations. Forexample, useful vectors may consist of segments of chromosomal,non-chromosomal or synthetic DNA sequences. The expression vectors ofthe invention are characterized by at least one expression controlsequence that may be operatively linked to a TRELL DNA sequence insertedin the vector, in order to control and to regulate the expression of theDNA sequence.

Furthermore, within each expression vector, various sites may beselected for insertion of a TRELL sequence of the invention. The sitesare usually designated by a restriction endonuclease which cuts them,and these sites and endonucleases are well recognized by those skilledin the art. It is of course to be understood that an expression vectoruseful in this invention need not have a restriction endonuclease sitefor insertion of the desired DNA fragment Instead, the vector may becloned to the fragment by alternate means. The expression vector, and inparticular the site chosen therein for insertion of a selected DNAfragment, and its operative linking therein to an expression controlsequence, is determined by a variety of factors. These factors include,but are not limited to, the size of the protein to be expressed, thesusceptibility of the desired protein to proteolytic degradation by hostcell enzymes, number of sites susceptible to a particular restrictionenzyme, contamination or binding of the protein to be expressed by hostcell proteins which may prove difficult to remove during purification.Additional factors which may be considered include expressioncharacteristics such as the location of start and stop codons relativeto the vector sequences, and other factors which will be recognized bythose skilled in the art. The choice of a vector and insertion site forthe claimed DNA sequences is determined by a balancing of these factors,not all selections being equally effective for a desired application.However, it is routine for one skilled in the art to analyze theseparameters and choose an appropriate system depending on the particularapplication.

One skilled in the art can readily make appropriate modifications to theexpression control sequences to obtain higher levels of proteinexpression, i.e. by substitution of codons, or selecting codons forparticular amino acids that are preferentially used by particularorganisms, to minimize proteolysis or to alter glycosylationcomposition. Likewise, cysteines may be changed to other amino acids tosimplify production, refolding or stability problems.

Thus, not all host/expression vector combinations function with equalefficiency in expressing the DNA sequences of this invention. However, aparticular selection of a host/expression vector combination may be madeby those of skill in the art. Factors one may consider include, forexample, the compatibility of the host and vector, toxicity to the hostof the proteins encoded by the DNA sequence, ease of recovery of thedesired protein, expression characteristics of the DNA sequences andexpression control sequences operatively linked to them, biosafety,costs and the folding, form or other necessary post-expressionmodifications of the desired protein.

The TRELL and homologs thereof produced by hosts transformed with thesequences of the invention, as well as native TRELL purified by theprocesses of this invention, or produced from the claimed amino acidsequences, are useful in a variety of compositions and methods foranticancer and immunoregulatory applications. They are also useful intherapy and methods directed to other diseases.

This invention also relates to the use of the DNA sequences disclosedherein to express TRELL under abnormal conditions, i.e. in a genetherapy setting. TRELL may be expressed in tumor cells under thedirection of promoters appropriate for such applications. Suchexpression could enhance anti-tumor immune responses or directly affectthe survival of the tumor. Cytokines such as TRELL can also affect thesurvival of an organ graft by altering the local immune response. Inthis case, the graft itself or the surrounding cells would be modifiedwith an engineered TRELL gene.

Another aspect of the invention relates to the use of the isolatednucleic acid encoding TRELL in “antisense” therapy. As used herein,“antisense” therapy refers to administration or in situ generation ofoligonucleotides or their derivatives which specifically hybridize undercellular conditions with the cellular mRNA and/or DNA encoding TRELL, soas to inhibit expression of the encoded protein, i.e. by inhibitingtranscription and/or translation. The binding may be by conventionalbase pair complementarity, or, for example, in the case of binding toDNA duplexes, through specific interactions in the major groove of thedouble helix. In general, “antisense” therapy refers to a range oftechniques generally employed in the art, and includes any therapy whichrelies on specific binding to oligonucleotide sequences.

An antisense construct of the present invention can be delivered, forexample, as an expression plasmid, which, when transcribed in the cell,produces RNA which is complementary to at least a portion of thecellular mRNA which encodes TRELL. Alternatively, the antisenseconstruct can be an oligonucleotide probe which is generated ex vivo.Such oligonucleotide probes are preferably modified oligonucleotideswhich are resistant to endogenous nucleases, and are therefor stable invivo. Exemplary nucleic acids molecules for use as antisenseoligonucleotides are phosphoramidates, phosphothioate andmethylphosphonate analogs of DNA (See, e.g., U.S. Pat. Nos. 5,176,996;5,264,564; and 5,256,775). Additionally, general approaches toconstructing oligomers useful in antisense therapy have been reviewed,for example, by Van Der Krol et al., (1988) Biotechniques 6:958-976; andStein et al. (1988) Cancer Res 48: 2659-2668, specifically incorporatedherein by reference.

C. TRELL AND ITS AMINO ACID SEQUENCES

The Tumor Necrosis Factor Family Related Protein (TRELL) of theinvention, as discussed above, is a member of the TNF family. Theprotein, fragments or homologs thereof may have wide therapeutic anddiagnostic applications.

TRELL is present in many tissues, in a pattern that is relatively uniqueamong members of the TNF family. Since members of the TNF family areinvolved in the regulation of a cell death and survival, and celldifferentiation, it is possible that TRELL is also involved in cellsurvival, differentiation, and repair in various tissues.

Although the precise three dimensional structure of TRELL is not known,it is predicted that, as a member of the TNF family, it may sharecertain structural characteristics with other members of the family.Both mouse and human TRELL are disclosed herein. Mouse TRELL, as deducedfrom the existing cDNA sequence, comprises a stretch of at least 21hydrophobic amino acids, which presumably acts as a membrane anchoringdomain for a type II membrane protein. The amino acid sequence of mTRELLis described in SEQ ID. NO. 2.

Human TRELL comprises an N-terminal hydrophilic cytoplasmic domain, aroughly 27 amino acid hydrophobic, transmembrane type II domain and a204 amino acid extracellular domain. The amino acid sequence of hTRELLis described in SEQ. ID. NO.:4.

FIG. 1 depicts an amino acid sequence comparison of human and mouseTRELL.

While a 52 amino acid N-terminal region can be predicted from an openreading frame in the cDNA clone, the exact starting methionine cannot bepredicted. Met-36 has a reasonable consensus Kozak sequence which maymake it the preferred starting codon. Comparison of the TRELL sequencewith other members of the human TNF family reveals considerablestructural similarity. For example, as can be seen in FIGS. 2A and 2B,all the proteins resemble Type II membrane proteins, and share severalregions of sequence conservation in the extracellular domain. Regionswith bars over the sequences indicate those sequences in TNF and LTαinvolved in a β strand organization of the molecules. Putative N-linkedglycosylation sites are underlined. Asterisks indicate the cysteinesinvolved in a disulfide linkage in TNF. The conserved domains are likelyto be involved in intersubunit interactions and sheet organization.

An EST search revealed a human clone of 345 bases which is highlyhomologous to the mouse TRELL. A human TRELL amino acid sequence is setforth in SEQ. ID. NO. 4. The open reading frames encoded by the EST donot contain the sequence motifs which would allow one to characterizethis sequence as a member of the TNF family of ligands, e.g. the motifused by Wiley et al. to identify a TRAIL EST within the existingdatabase.

The novel polypeptides of the invention specifically interact with areceptor, which has not yet been identified. However, the peptides andmethods disclosed herein enable the identification of receptors whichspecifically interact with TRELL or fragments thereof.

The claimed invention in certain embodiments includes peptides derivedfrom TRELL which have the ability to bind with TRELL receptors.Fragments of TRELL can be produced in several ways, e.g., recombinantly,by PCR, proteolytic digestion or by chemical synthesis. Internal orterminal fragments of a polypeptide can be generated by removing one ormore nucleotides from one end or both ends of a nucleic acid whichencodes the polypeptide. Expression of the mutagenized DNA producespolypeptide fragments. Digestion with “end-nibbling” endonucleases canthus generate DNA's which encode a variety of fragments. DNA's whichencode fragments of a protein can also be generated by random shearing,restriction digestion or a combination of the above discussed methods.

Polypeptide fragments can also be chemically synthesized usingtechniques known in the art such as conventional Merrifield solid phasef-moc or t-boc chemistry. For example, peptides and DNA sequences of thepresent invention may be arbitrarily divided into fragments of desiredlength with no overlap of the fragment, or divided into overlappingfragments of a desired length. Methods such as these are described inmore detail below.

D. GENERATION OF SOLUBLE TRELL

Soluble forms of the ligand can often signal effectively and hence canbe administered as a drug which now mimics the natural membrane form. Itis possible that TRELL is naturally secreted as a soluble cytokine,however, if it is not, one can reengineer the gene to force secretion.To create a soluble secreted form of TRELL, one would remove at the DNAlevel the N-terminus transmembrane regions, and some portion of thestalk region, and replace them with a type I leader or alternatively atype II leader sequence that will allow efficient proteolytic cleavagein the chosen expression system. A skilled artisan could vary the amountof the stalk region retained in the secretion expression construct tooptimize both receptor binding properties and secretion efficiency. Forexample, the constructs containing all possible stalk lengths, i.e.N-terminal truncations, could be prepared such that proteins starting atamino acids 81 to 139 would result The optimal length stalk sequencewould result from this type of analysis.

E. GENERATION OF ANTIBODIES REACTIVE WITH TRELL

The invention also includes antibodies specifically reactive with TRELLor its receptor. Anti-protein/anti-peptide antisera or monoclonalantibodies can be made by standard protocols (See, for example,Antibodies: A Laboratory Manual ed. by Harlow and Lane (Cold SpringHarbor Press: 1988)). A mammal such as a mouse, a hamster or rabbit canbe immunized with an immunogenic form of the peptide. Techniques forconferring immunogenicity on a protein or peptide include conjugation tocarriers, or other techniques, well known in the art.

An immunogenic portion of TRELL or its receptor can be administered inthe presence of an adjuvant. The progress of immunization can bemonitored by detection of antibody titers in plasma or serum. StandardELISA or other immunoassays can be used with the immunogen as antigen toassess the levels of antibodies.

In a preferred embodiment, the subject antibodies are immunospecific forantigenic determinants of TRELL or its receptor, e.g. antigenicdeterminants of a polypeptide of SEQ ID NO: 2 or 4, or a closely relatedhuman or non-human mammalian homolog (e.g. 70, 80 or 90 percenthomologous, more preferably at least 95 percent homologous). In yet afurther preferred embodiment of the present invention, the anti-TRELL oranti-TRELL-receptor antibodies do not substantially cross react (i.e.react specifically) with a protein which is e.g., less than 80 percenthomologous to SEQ ID NO 2 or 4; preferably less than 90 percenthomologous with SEQ ID NO: 2 or 4; and, most preferably less than 95percent homologous with SEQ ID NO: 2 or 4. By “not substantially crossreact”, it is meant that the antibody has a binding affinity for anon-homologous protein which is less than 10 percent, more preferablyless than 5 percent, and even more preferably less than 1 percent, ofthe binding affinity for a protein of SEQ ID NO 2 or 4.

The term antibody as used herein is intended to include fragmentsthereof which are also specifically reactive with TRELL orTRELL-receptor. Antibodies can be fragmented using conventionaltechniques and the fragments screened for utility in the same manner asdescribed above for whole antibodies. For example, F(ab′)₂ fragments canbe generated by treating antibody with pepsin. The resulting F(ab′)₂fragment can be treated to reduce disulfide bridges to produce Fab′fragments. The antibodies of the present invention are further intendedto include biospecific and chimeric molecules having anti-TRELL oranti-TRELL-receptor activity. Thus, both monoclonal and polyclonalantibodies (Ab) directed against TRELL and its receptor, and antibodyfragments such as Fab′ and F(ab′)₂, can be used to block the action ofTRELL and its receptor.

Various forms of antibodies can also be made using standard recombinantDNA techniques. (Winter and Milstein, Nature 349: 293-299 (1991)specifically incorporated by reference herein.) For example, chimericantibodies can be constructed in which the antigen binding domain froman animal antibody is linked to a human constant domain (e.g. Cabilly etal., U.S. Pat. No. 4,816,567, incorporated herein by reference).Chimeric antibodies may reduce the observed immunogenic responseselicited by animal antibodies when used in human clinical treatments.

In addition, recombinant “humanized antibodies” which recognize TRELL orits receptor can be synthesized. Humanized antibodies are chimerascomprising mostly human IgG sequences into which the regions responsiblefor specific antigen-binding have been inserted. Animals are immunizedwith the desired antigen, the corresponding antibodies are isolated, andthe portion of the variable region sequences responsible for specificantigen binding are removed. The animal-derived antigen binding regionsare then cloned into the appropriate position of human antibody genes inwhich the antigen binding regions have been deleted. Humanizedantibodies minimize the use of heterologous (i.e. inter species)sequences in human antibodies, and thus are less likely to elicit immuneresponses in the treated subject.

Construction of different classes of recombinant antibodies can also beaccomplished by making chimeric or humanized antibodies comprisingvariable domains and human constant domains (CH1, CH2, CH3) isolatedfrom different classes of immunoglobulins. For example, antibodies withincreased antigen binding site valencies can be recombinantly producedby cloning the antigen binding site into vectors carrying the human:chain constant regions. (Arulanandam et al., J. Exp. Med., 177:1439-1450 (1993), incorporated herein by reference.)

In addition, standard recombinant DNA techniques can be used to alterthe binding affinities of recombinant antibodies with their antigens byaltering amino acid residues in the vicinity of the antigen bindingsites. The antigen binding affinity of a humanized antibody can beincreased by mutageneesis based on molecular modeling. (Queen et al.,Proc. Natl. Acad. Sci. 86: 10029-33 (1989) incorporated herein byreference.

F. GENERATION OF ANALOGS Production of Altered DNA and Peptide Sequences

Analogs of TRELL can differ from the naturally occurring TRELL in aminoacid sequence, or in ways that do not involve sequence, or both.Non-sequence modifications include in vivo or in vitro chemicalderivatization of TRELL. Non-sequence modifications include, but are notlimited to, changes in acetylation, methylation, phosphorylation,carboxylation or glycosylation.

Preferred analogs include TRELL or biologically active fragmentsthereof, whose sequences differ from the sequence given in SEQ ID NOS. 2and 4, by one or more conservative amino acid substitutions, or by oneor more non-conservative amino acid substitutions, deletions orinsertions which do not abolish the activity of TRELL. Conservativesubstitutions typically include the substitution of one amino acid foranother with similar characteristics, e.g. substitutions within thefollowing groups: valine, glycine; glycine, alanine; valine, isoleucine,leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine,threonine; lysine, arginine; and, phenylalanine, tyrosine.

TABLE 1 CONSERVATIVE AMINO ACID REPLACEMENTS for amino Acid code replacewith any of: Alanine A D-Ala, Gly, Beta-Ala, L-Cys, D-Cys Arginine RD-Arg, Lys, D-Lys, homo-Arg, D-homo-Arg, Met, He, D-Met, D-Ile, Orn,D-Orn Asparagine N D-Asn, Asp, D-Asp, Glu, D-Glu, Gin, D-Gln AsparticAcid D D-Asp, D-Asn, Asn, Glu, D-Glu, Gin, D-Gln Cysteine C D-Cys,S-Me-Cys, Met, D-Met, Thr, D-Thr Glutamine Q D-Gln, Asn, D-Asn, Glu,D-Glu, Asp, D-Asp Glutamic Acid E D-Glu, D-Asp, Asp, Asn, D-Asn, Gin,D-Gln Glycine G Ala, D-Ala, Pro, D-Pro, -Ala, Acp Isoleucine I D-Ile,Val, D-Val, Leu, D-Leu, Met, D-Met Leucine L D-Leu, Val, D-Val, Leu,D-Leu, Met, D-Met Lysine K D-Lys, Arg, D-Arg, Homo-arg, D-homo-Arg, Met,D-Met, Ile, D-Ile, Orn, D-Orn Methionine M D-Met, S-Me-Cys, Ile, D-Ile,Leu, D-Leu, Val, D-Val Phenylalanine F D-Phe, Tyr, D-Thr, L-Dopa, His,D-His, Trp, D-Trp, Trans-3,4 or 5-phenylproline, cis-3,4, or5-phenylproline Proline P D-Pro, L-I-thoazolidine-4-carboxylic acid,D-or L-1-oxazolidine-4-carboxylic acid Serine S D-Ser, Thr, D-Thr,allo-Thr, Met, D-Met, Met(O), D-Met(O), L-Cys, D-Cys Threonine T D-Thr,Ser, D-Ser, allo-Thr, Met, D-Met, Met(O), D-Met(O), Val, D-Val TyrosineY D-Tyr, Phe, D-Phe, L-Dopa, His, D-His Valine V D-Val, Leu, D-Leu, Ile,D-Ile, Met, D-Met

Useful methods for mutagenesis include PCR mutagenesis and saturationmutagenesis as discussed in more detail below. A library of random aminoacid sequence variants can also be generated by the synthesis of a setof degenerate oligonucleotide sequences.

PCR Mutagenesis

In PCR mutagenesis, reduced Taq polymerase fidelity can be used tointroduce random mutations into a cloned fragment of DNA (Leung et al.,1989, Technique 1:11-15). This is a very powerful and relatively rapidmethod of introducing random mutations. The DNA region to be mutagenizedcan be amplified using the polymerase chain reaction (PCR) underconditions that reduce the fidelity of DNA synthesis by Taq DNApolymerase, e.g., by using a dGTP/dATP ratio of five and adding Mn²+ tothe PCR reaction. The pool of amplified DNA fragments can be insertedinto appropriate cloning vectors to provide random mutant libraries.

Saturation Mutagenesis

Saturation mutagenesis allows for the rapid introduction of a largenumber of single base substitutions into cloned DNA fragments (Mayers etal., 1985, Science 229:242). This technique includes generation ofmutations, e.g., by chemical treatment or irradiation of single-strandedDNA in vitro, and synthesis of a complimentary DNA strand. The mutationfrequency can be modulated by modulating the severity of the treatment,and essentially all possible base substitutions can be obtained. Becausethis procedure does not involve a genetic selection for mutant fragmentsboth neutral substitutions, as well as of a protein can be prepared byrandom mutagenesis of DNA which those that alter function, can beobtained. The distribution of point mutations is not biased towardconserved sequence elements.

Degenerate Oligonucleotides

A library of homologs can also be generated from a set of degenerateoligonucleotide sequences. Chemical synthesis of degenerate sequencescan be carried out in an automatic DNA synthesizer, and the syntheticgenes then ligated into an appropriate expression vector. The synthesisof degenerate oligonucleotides is known in the art^(xxxi) Suchtechniques have been employed in the directed evolution of otherproteins^(xxxii).

Non-random or directed, mutagenesis techniques can be used to providespecific sequences or mutations in specific regions. These techniquescan be used to create variants which include, e.g., deletions,insertions, or substitutions, of residues of the known amino acidsequence of a protein. The sites for mutation can be modifiedindividually or in series, e.g., by (1) substituting first withconserved amino acids and then with more radical choices depending uponresults achieved, (2) deleting the target residue, or (3) insertingresidues of the same or a different class adjacent to the located site,or combinations of options 1-3.

Alanine Scanning Mutagenesis

Alanine scanning mutagenesis is a useful method for identification ofcertain residues or regions of the desired protein that are preferredlocations or domains for mutagenesis, Cunningham and Wells (Science244:1081-1085, 1989) specifically incorporated by reference. In alaninescanning, a residue or group of target residues are identified (e.g.,charged residues such as Arg, Asp, His, Lys, and Glu) and replaced by aneutral or negatively charged amino acid (most preferably alanine orpolyalanine). Replacement of an amino acid can affect the interaction ofthe amino acids with the surrounding aqueous environment in or outsidethe cell. Those domains demonstrating functional sensitivity to thesubstitutions can then be refined by introducing further or othervariants at or for the sites of substitution. Thus, while the site forintroducing an amino acid sequence variation is predetermined, thenature of the mutation per se need not be predetermined. For example, tooptimize the performance of a mutation at a given site, alanine scanningor random mutagenesis may be conducted at the target codon or region andthe expressed desired protein subunit variants are screened for theoptimal combination of desired activity.

Oligonucleotide-Mediated Mutagenesis

Oligonucleotide-mediated mutagenesis is a useful method for preparingsubstitution, deletion, and insertion variants of DNA, see, e.g.,Adelman et al., (DNA 2:183, 1983) incorporated herein by reference.Briefly, the desired DNA can be altered by hybridizing anoligonucleotide encoding a mutation to a DNA template, where thetemplate is the single-stranded form of a plasmid or bacteriophagecontaining the unaltered or native DNA sequence of the desired protein.After hybridization, a DNA polymerase is used to synthesize an entiresecond complementary strand of the template that will thus incorporatethe oligonucleotide primer, and will code for the selected alteration inthe desired protein DNA. Generally, oligonucleotides of at least 25nucleotides in length are used. An optimal oligonucleotide will have 12to 15 nucleotides that are completely complementary to the template oneither side of the nucleotide(s) coding for the mutation. This ensuresthat the oligonucleotide will hybridize properly to the single-strandedDNA template molecule. The oligonucleotides are readily synthesizedusing techniques known in the art such as that described by Crea et al.(Proc. Nad. Acad. Sci. USA, 75: 5765[1978]) incorporated herein byreference.

Cassette Mutagenesis

Another method for preparing variants, cassette mutagenesis, is based onthe technique described by Wells et al. (Gene, 34:315[1985])incorporated herein by reference. The starting material can be a plasmid(or other vector) which includes the protein subunit DNA to be mutated.The codon(s) in the protein subunit DNA to be mutated are identified.There must be a unique restriction endonuclease site on each side of theidentified mutation site(s). If no such restriction sites exist, theymay be generated using the above-described oligonucleotide-mediatedmutagenesis method to introduce them at appropriate locations in thedesired protein subunit DNA. After the restriction sites have beenintroduced into the plasmid, the plasmid is cut at these sites tolinearize it. A double-stranded oligonucleotide encoding the sequence ofthe DNA between the restriction sites but containing the desiredmutation(s) is synthesized using standard procedures. The two strandsare synthesized separately and then hybridized together using standardtechniques. This double-stranded oligonucleotide is referred to as thecassette. This cassette is designed to have 3′ and 5′ ends that arecomparable with the ends of the linearized plasmid, such that it can bedirectly ligated to the plasmid. This plasmid now contains the mutateddesired protein subunit DNA sequence.

Combinatorial Mutagenesis

Combinatorial mutagenesis can also be used to generate mutants. E.g.,the amino acid sequences for a group of homologs or other relatedproteins are aligned, preferably to promote the highest homologypossible. All of the amino acids which appear at a given position of thealigned sequences can be selected to create a degenerate set ofcombinatorial sequences. The variegated library of variants is generatedby combinatorial mutagenesis at the nucleic acid level, and is encodedby a variegated gene library. For example, a mixture of syntheticoligonucleotides can be enzymatically ligated into gene sequences suchthat the degenerate set of potential sequences are expressible asindividual peptides, or alternatively, as a set of larger fusionproteins containing the set of degenerate sequences.

Various techniques are known in the art for screening generated mutantgene products. Techniques for screening large gene libraries ofteninclude cloning the gene library into replicable expression vectors,transforming appropriate cells with the resulting library of vectors,and expressing the genes under conditions in which detection of adesired activity, e.g., in this case, binding to TRELL or its receptor,facilitates relatively easy isolation of the vector encoding the genewhose product was detected. Each of the techniques described below isamenable to high through-put analysis for screening large numbers ofsequences created, e.g., by random mutagenesis techniques.

The invention also provides for reduction of the protein binding domainsof the subject TRELL polypeptides or their receptors, to generatemimetics, e.g. peptide or non-peptide agents. The peptide mimetics areable to disrupt binding of a TRELL and its receptor. The criticalresidues of TRELL involved in molecular recognition of a receptorpolypeptide or of a downstream intracellular protein, can be determinedand used to generate TRELL or its receptor-derived peptidomimetics whichcompetitively or noncompetitively inhibit binding of the TRELL with areceptor. (see, for example, “Peptide inhibitors of human papillomavirus protein binding to retinoblastoma gene protein” European patentapplications EP-412,762A and EP-531,080A, specifically incorporatedherein by reference.

G. PHARMACEUTICAL COMPOSITIONS

By making available purified and recombinant-TRELL, the presentinvention provides assays which can be used to screen for drugscandidates which are either agonists or antagonists of the normalcellular function, in this case, of TRELL or its receptor. In oneembodiment, the assay evaluates the ability of a compound to modulatebinding between TRELL and its receptor. A variety of assay formats willsuffice and, in light of the present inventions, will be comprehended bythe skilled artisan.

In many drug screening programs which test libraries of compounds andnatural extracts, high throughput assays are desirable in order tomaximize the number of compounds surveyed in a given period of time.Assays which are performed in cell-free systems, such as may be derivedwith purified or semi-purified proteins, are often preferred as“primary” screens in that they can be generated to permit rapiddevelopment and relatively easy detection of an alteration in amolecular target which is mediated by a test compound. Moreover, theeffects of cellular toxicity and/or bioavailability of the test compoundcan be generally ignored in the in vitro system, the assay instead beingfocused primarily on the effect of the drug on the molecular target asmay be manifest in an alteration of binding affinity with other proteinsor change in enzymatic properties of the molecular target.

Pharmaceutical compositions of the invention may comprise atherapeutically effective amount of TRELL or TRELL-receptor, orfragments or mimetics thereof, and, optionally may includepharmaceutically acceptable carriers. Accordingly, this inventionprovides methods for treatment of cancer, and methods of stimulating, orin certain instances, inhibiting the immune system, or parts thereof byadministering a pharmaceutically effective amount of a compound of theinvention or its pharmaceutically acceptable salts or derivatives. Itshould of course by understood that the compositions and methods of thisinvention can be used in combination with other therapies for varioustreatments.

The compositions can be formulated for a variety of routes ofadministration, including systemic, topical or localized administration.For systemic administration, injection is preferred, includingintramuscular, intravenous, intraperitoneal, and subcutaneous forinjection, the compositions of the invention can be formulated in liquidsolutions, preferably in physiologically compatible buffers such asHank's solution or Ringer's solution. In addition, the compositions maybe formulated in solid form and, optionally, redissolved or suspendedimmediately prior to use. Lyophilized forms are also included in theinvention.

The compositions can be administered orally, or by transmucosal ortransdermal means. For transmucosal or transdermal administration,penetrants appropriate to the barrier to be permeated are used in theformulation. Such penetrants are known in the art, and include, forexample, for transmucosal administration, bile salts, fusidic acidderivatives, and detergents. Transmucosal administration may be throughnasal sprays or using suppositories. For oral administration, thecompositions are formulated into conventional oral administration formssuch as capsules, tablets, and tonics. For topical administration, thecompositions of the invention are formulated into ointments, salves,gels, or creams as known in the art.

Preferably the compositions of the invention will be in the form of aunit dose and will be administered one or more times a day. The amountof active compound administered at one time or over the course oftreatment will depend on many factors. For example, the age and size ofthe subject, the severity and course of the disease being treated, themanner and form of administration, and the judgments of the treatingphysician. However, an effective dose may be in the range of from about0.005 to about 5 mg/kg/day, preferably about 0.05 to about 0.5mg/kg/day. One skilled in the art will recognize that lower and higherdoses may also be useful.

Gene constructs according to the invention can also be used as a part ofa gene therapy protocol to deliver nucleic acids encoding either anagonistic or antagonistic form of a TRELL polypeptide.

Expression constructs of TRELL can be administered in any biologicallyeffective carrier, e.g., any formulation or composition capable ofeffectively delivering the gene for TRELL to cells in vivo. Approachesinclude insertion of the gene in viral vectors which can transfect cellsdirectly, or delivering plasmid DNA with the help of, for example,liposomes, or intracellular carriers, as well as direct injection of thegene construct. Viral vector transfer methods are preferred.

A pharmaceutical preparation of the gene therapy construct can consistessentially of the gene delivery system in an acceptable diluent, or cancomprise a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery system can beproduced intact from recombinant cells, e.g. retroviral vectors, thepharmaceutical preparation can comprise one or more cells which producethe gene delivery system.

In addition to use in therapy, the oligomers of the invention may beused as diagnostic reagents to detect the presence or absence of thetarget DNA, RNA or amino acid sequences to which they specifically bind.In other aspects, the claimed invention may be used to evaluate achemical entity for its ability to interact with, e.g., bind orphysically associate with a TRELL polypeptide, or fragment thereof. Themethod includes contacting the chemical entity with the TRELLpolypeptide, and evaluating the ability of the entity to interact withthe TRELL. Additionally, the TRELL of the invention can be used inmethods of evaluating naturally occurring ligands or receptors of TRELL,as well as to evaluate chemical entities which associate or bind withreceptors of TRELL.

In certain aspects, the claimed invention features a method forevaluating a chemical entity for the ability to modulate the interactionbetween TRELL and its receptor. The method includes combining a TRELLreceptor, and TRELL under conditions wherein the pair is capable ofinteracting, adding the chemical entity to be evaluated and detectingthe formation or dissolution of complexes. These modulating agents maybe further evaluated in vitro, e.g. by testing its activity in a cellfree system, and then, optionally administering the compound to a cellor animal, and evaluating the effect

H. EXAMPLES 1. Isolation of TRELL cDNAS

a) Cloning of Murine TRELL

The cDNA coding for mTRELL was isolated by PCR from a cDNA library frommouse peritoneal macrophages. The amino acid sequence and the placementof the transmembrane region are typical of a membrane protein. The aminoacid sequence of mTRELL is set forth in SEQ. ID. NO. 2, and the DNAsequence is set forth in SEQ. ID. NO. 1.

Macrophage cells were obtained from Balb/c mice by peritoneal lavage andcells that adhered to plastic within one hour were lysed and processedfor RNA extraction. An antisense oligonucleotide primer5′GTTCCAGGCCAGCCTGGG3′ (SEQ ID NO: 5) from a mouse erythropoietinsequence was synthesized. C. B. Shoemaker and L. D. Mistock, “Murineerythropoietin gene: cloning, expression and human gene homology”, Mol.Cell. Biol., 6, 849 (1986), specifically incorporated herein byreference. This primer was used in a 5′ RACE protocol following therecommendation of the manufacturer (5′ RACE system from BRL) inassociation with the BRL-designed anchor primer. A first strand of cDNAwas made from RNA from one hour adherent peritoneal macrophages.Amplification was done in a Perkin Elmer DNA thermal cycler with Taq DNApolymerase from Perkin Elmer. After a denaturation of 5 min. at 94° C.,cycling conditions were as follows: 35 cycles at 94° C. for 30 sec., 55°C. for 30 sec. and 72° C. for 3 min. An additional extension at 72° C.was performed and then reactions were held at 4° C. Analysis of the PCRexperiment on agarose gel revealed 2 amplified fragments of 650 bp and500 bp. The 2 fragments were excised from the gel, inserted in pBS-Tvectors and sequenced. The two inserts were different. They both had ateach extremity the same erythropoietin gene specific oligonucleotideused to prime the PCR synthesis. Northern hybridizations with 32Plabeled-random-primed fragments indicated that they hybridized to twodifferent RNA, the 500 bp fragment hybridizing to a 1.4 kb RNA inmacrophages. ³²P-labeled-riboprobes in both directions were used inNorthern hybridization to determine the orientation of the cDNA.

From the determined orientations and sequences, two internal primers forthe 1.4 Kb mRNA were derived. These were used in 3′ and 5′ RACE-PCRrespectively. The 3′RACE experiment revealed a 750 bp fragment which wasinserted in a pBS-T vector and sequenced. It corresponds to the 3′ endof the 1.4 Kb RNA since the sequence possess a polyA addition signalAATAAA (SEQ ID NO: 6) just prior to the poly A tract. The 5′RACE did notreveal any band. The Clontech Marathon cDNA amplification kit was usedto prepare a cDNA library from one hour adherent macrophage. A 1040 bpPCR fragment, isolated by PCR with sense and antisense oligonucleotideprimers from the determined cDNA sequence were used, and the universalprimer from the kit. This resulted in the isolation of a fragment of alarger size than the original 1040 bp fragment. The new fragment whichwas sequenced added 60 bp to the 5′sequence (SEQ ID NO: 1).

RNA were extracted from mouse thioglycolate induced peritonealmacrophages after 1 hour adherence. Hybridization was performed with³²P-labeled mTRELL cDNA. FIG. 3 depicts northern analysis of TRELL mRNAexpression in mouse peritoneal macrophages and in different mousetissues.

The first 21 amino acids delineate a hydrophobic, transmembrane domain.No identical sequences at the nucleotide or the amino acid levels werefound in the available databases. Using the PROSITE program, and the 225amino acid sequence it was determined that the sequence belonged to theTNF family of proteins. The protein also possessed the different domainsdescribed for LT-□ and other members of this family (J. Browning et al.,Lymphotoxin-□, a novel member of the TNF family that forms a heteromericcomplex with lymphotoxin on the cell surface”, Cell, 72, 847 (1993); C.F. Ware et al., “The ligands and receptors of the lymphotoxin system”,in Pathways for cytolysis, G. M. Griffiths and J. Tschopp (Eds),Springer-Verlag, Berlin, Heidelberg, p 175-218 (1995), each of which isspecifically incorporated herein by reference). This sequence is unique.At the nucleotide or amino acid levels, weak identity or similarity wereobserved with the different members of the TNF family or with anysequences. Searching in EST data bases, 1 human sequence was clearlyhomologous to the murine sequence. The clone 154742, 5′ (GenBankaccession no: R55379) from a breast library made by Soares, WashingtonUniversity, has a 345 base pair sequence, 89% homologous to the murineTRELL. No human sequence in the available databases was found matchingthe available 5′ DNA of mTRELL.

b) Cloning of Human TRELL

i) Generation of Oligonucleotide Probes and PCR Primers.

The sequence of the human EST R55379 which has homology to mouse TRELLwas used as a basis for synthesis of oligonucleotide primers. Two sensestrand 20mer oligonucleotides:

LTB-065 5=-CCC TGC GCT GCC TGG AGG AA (NT 70-89 of R55379) (SEQ ID NO:7)

LTB-066 5=-TGA TGA GGG GAA GGC TGT CT (NT 14-33 of R55379) (SEQ ID NO:8) and one antisense 20mer oligonucleotide:

LTB-067 5=-AGA CCA GGG CCC CTC AGT GA (NT 251-270 of R55379) (SEQ ID NO:9) were synthesized.

ii) Identification of mRNA and cDNA Library Source for Cloning hTRELL.

PolyA+ mRNA from Human liver (cat#6510-1), spleen (cat#6542-1) and lymphnode (cat#6594-1) were purchased from Clontech. PolyA+ mRNA from Humancell lines THP-1, U937 and 11-23 were generated at Biogen, Cambridge,Mass. A human tonsil cDNA library in Lambda gt10, and DNA from theTonsil library were also prepared at Biogen.

RT-PCR was performed on the six RNA samples. Each cDNA reactioncontained lug polyA+ mRNA, 50 mM Tris pH 8.3, 75 mM KCl, 3 mM MgCl₂, 10mM DTT, 250 uM dNTP, 50 ng random hexamer (50 ng/ul) and 400 unitsSuperscriptII™ Reverse transcriptase (Gibco BRL cat #6542-1) in a finalvolume of 40 ul. The reaction was incubated at 20 EC for 20 minutes, 42EC for 50 min., and 99 EC for 5 min. For PCR, one-fifth of each cDNAreaction or 100-1000 ng of the cDNA library DNA was used. Two PCRreactions for each sample were set up, one with primer pair LTB-065 andLTB-067 which yields a 201 bp PCR product, and the second reaction withprimer pair LTB-066 and LTB-067 which yields a 257 bp product if thetranscript is represented in the sample. PCR reactions were performed in10 mM Tris pH 8.3, 50 mM KCl, 1.5 mM MgCl₂, 0.01% gelatin, 10% DMSO 100uM dNTP, 30 pmole each primer and 5 units Amplitaq™ (Perkin Elmer cat#N801-0060). PCR was carried out in a Perkin Elmer Cetus DNA ThermalCycler Model #480. Cycle conditions used were 95 EC 1 minute, 60 EC 1minute, and 72 EC 1 minute for 35 cycles.

The correct size products were obtained from liver, spleen, lymph node,THP-1 and tonsil, but not from U937 or II-23 mRNA. The 201 bp PCRproduct generated from liver was purified for use as a probe forscreening the cDNA library.

iii) cDNA Library Screening

Having demonstrated by PCR that the tonsil library contained TRELL, onemillion plaque forming units (PFU) from the Lambda gtl10 Human tonsilcDNA library were plated at a density of 1×10⁵ PFU/Nunc™ plate.Duplicate lifts were made onto 20×20 cm Schleicher and Scheull BA-S 85Optitran™ filters. The 201 bp PCR product was ³²P labeled by randompriming (Feinberg and Vogelstein, Anal. Biochem 137:266-267, 1984specifically incorporated herein by reference). The filters werehybridized overnight at 65 EC in 400 ml plaque screen buffer (50 mM TrispH 7.5, 1 M NaCl, 0.1% sodium pyrophosphate, 0.2% PVP and 0.2% Ficoll)containing 10% dextran sulphate, 100 μg/ml tRNA and 6×10⁵ CPM/ml probe.They were washed twice with plaque screen buffer and twice with 2×SSC,0.1% SDS at 65 C and exposed to film at −70 C. with an intensifyingscreen for 40 hours.

Duplicate positives were cored from the master plates into SM (100 mMNaCl, 10 mM MgSO₄, 50 mM Tris pH 7.5) plus gelatin. 12 of the positiveswere plaque purified. Lambda miniprep DNA from 12 purified candidateswas digested with Not1, electrophoresed on 1% agarose gel, Southernblotted and hybridized with the 201 bp probe. The clones with thelargest inserts (approximately 2 kb) which hybridized to the probe wereselected for large scale DNA purification and DNA sequencing. Theinserts from each of these clones was subcloned into the Not1 site ofpBluescript SK+ ™ (Strategene #212205). DNA sequence was obtained fromthe Lambda DNA and the plasmid DNA. Clone Fla which has an cDNA insertof 2006 bp appeared to have an intron in the 5′ end of the coding regionand did not contain a complete open reading frame. Clone A2a, alsocalled PB133 contained a cDNA insert of 1936 bp. This clone contained543 bp 5′ untranslated region, an open reading frame of 852 bp and 3′untransated region but no polyadenylation signal or polyA tail.

The nucleotide sequence encoding the open reading frame of the hTRELLcDNA clone A2a is set forth in SEQ ID. NO. 3. The deduced 284 amino acidsequence is set forth in SEQ ID. NO. 4. The second methionine atposition 36 may be a more likely translation start site, since this sitemore closely meets the requirements for a start as defined by Kozak.

Using the sequences identified, the sequences of cDNAs coding on TRELLwere determined. From the DNA sequences described above (i.e. SEQ. ID.NO. 3), we deduced the amino acid sequences of TRELL (SEQ. ID. NO. 4).It should be clear that given the current state of theprotein-engineering art, an artisan could make purposeful alterations,insertions or deletions in these amino acid sequences and obtain avariety of molecules having substantially the same biological orimmunological activities as those of the molecules we have describedherein.

iv) Northern Analysis of Human TRELL Expression

A 440 bp PpuM1/BstX1 fragment of the human cDNA clone 2a was 32P labeledby random priming and used to probe commercial northern blots containingRNA from various human tissues. Northern analysis showed that the hTRELLfragment hybridized to a single mRNA species about 1.4 to 1.6 kb inlength. Human TRELL is expressed in most organs of the immune system,i.e. spleen, peripheral blood lymphocytes (pb1), lymph nodes, appendixbut was relatively low in thymus, fetal liver (source of progenitorlymphocytes) and bone marrow (FIG. 4). Therefore, organs of thesecondary immune system primarily express TRELL. Expression was alsodetected in the ovary, prostate, small intestine, colon, heart, brain,placenta, lung, liver, skeletal muscle, kidney and pancreas. Expressionwas relatively low in testis, liver, kidney and thymus. This patternindicates widespread expression closely resembling that of the TRAILligand except that TRAIL is poorly expressed in heart and brain.

c) Isolation of a Receptor Binding to the TRELL Ligand

Ligands of the TNF family can be used to identify and clone receptors.With the described TRELL sequence, one could fuse the 5′end of theextracellular domain of TRELL ligand which constitutes the receptorbinding sequence to a marker or tagging sequence and then add a leadersequence that will force secretion of the ligand in any of a number ofexpression systems. One example of this technology is described byBrowning et al., (1996) (JBC 271, 8618-8626) where the LT-β ligand wassecreted in such a form. The VCAM leader sequence was coupled to a shortmyc peptide tag followed by the extracellular domain of the LT-β. TheVCAM sequence is used to force secretion of the normally membrane boundLT-β molecule. The secreted protein retains a myc tag on the N-terminuswhich does not impair the ability to bind to a receptor. Such a secretedprotein can be expressed in either transiently transfected Cos cells ora similar system, e.g., EBNA derived vectors, insect cell/baculovirus,picchia etc. The unpurified cell supernatant can be used as a source ofthe tagged ligand.

Cells expressing the receptor can be identified by exposing them to thetagged ligand. Cells with bound ligand are identified in a FACSexperiment by labelling the myc tag with an anti-myc peptide antibody(9E10) followed by phycoerythrin (or a similar label) labelledanti-mouse immunoglobulin. FACS positive cells can be readily identifiedand would serve as a source of RNA encoding for the receptor. Anexpression library would then be prepared from this RNA via standardtechniques and separated into pools. Pools of clones would betransfected into a suitable host cell and binding of the tagged ligandto receptor positive transfected cells determined via microscopicexamination, following labelling of bound myc peptide tag with an enzymelabelled anti-mouse Ig reagent, i.e. galactosidase, alkaline phosphataseor luciferase labelled antibody. Once a positive pool has beenidentified, the pool size would be reduced until the receptor encodingcDNA is identified. This procedure could be carried out with either themouse or human TRELL as one may more readily lead to a receptor.

2. Cells and Reagents

All cells were obtained from the American Type Culture Collection (ATCC,Rockville, Md.) except for WEHI 164 clone 13 which was obtained from Dr.Kawashima (Geneva Biomedical Research Institute, Geneva, Switzerland).The HT29 subclone (HT29-14) was previously described (Browning et al.,1996) and the TNF sensitive ME180 subclone was obtained from Dr. CarlWare. The II-23 T cell hybridoma has been described (Browning et al.,1991). Balb/c mice were injected intraperitoneally 3 days beforesacrifice with 1.5 ml of thioglycolate broth (Difco Lab., MI). Cellswere taken from the peritoneal cavity and cultured at 10⁶ cells/ml for 1hr in DMEM (Gibco Lab). Non adherent cells were washed off the platesand the adherent cells, almost exclusively macrophages, were lysed inTri-Reagent (Molecular Research Center Inc.) and processed for RNAextraction.

Recombinant human TNF, LTa, LTa1/b2, antibodies to these proteins andthe receptor-Ig fusion proteins have been described previously (Browninget al., 1995). The anti-CD40L antibody 5C8 has been described. Apolyclonal anti-hTRELL serum was prepared by intra lymph node injectionof pure recombinant hTRELL in CFA as described previously (Browning andRibolini, 1989). After 2 months, an anti-hTRELL response was observedand immunoglobulin was purified using Protein A-Sepharose.

Mouse TRELL Cloning

The antisense oligonucleotide primer 5′GTTCCAGGCCAGCCTGGG3′ (SEQ ID NO:10) from the mouse erythropoietin sequence was used it in a 5′ RACEprotocol following the recommendation of the manufacturer (5′ RACEsystem from BRL) in association with the BRL-designed anchor primer.First strand cDNA was made from RNA from 1 hr. adherent peritonealmacrophages. Amplification was done in a Perkin Elmer DNA thermal cyclerwith Taq DNA polymerase. After a denaturation of 5 min. at 94/C, cyclingconditions were as follows: 35 cyles of 30 sec. at 94/C, 30 sec at 55/Cand 3 min at 72/C followed by a terminal additional extension at 72/C.Analysis of the PCR experiment on agarose gel revealed 2 amplifiedfragments of 650 bp and 500 bp. The 2 fragments were excised from thegel, inserted in pBS-T vectors and sequenced. Northern hybridizationswith ³²P labeled-random-primed fragments indicated that the 500 bpfragment hybridizing to a 1.4 kb RNA in macrophages. To determine theorientation of the cDNA, 32P-labeled-riboprobes in both direction wereused in Northern hybridization. From the determined orientations andsequences, we derived two internal primers for the 1.4 kb mRNA: 5′TCAGGTGCACTTTGATGAGG 3′ (SEQ ID NO: 11) and 5′CTGTCAGCTCCTCCTGAG 3′ (SEQID NO: 12) which were used in 3′ and 5′RACE-PCR respectively. The 3′RACEexperiment revealed a 750 bp fragment which was inserted in a pBS-Tvector and sequenced. It corresponded to the 3′ end of the 1.4 kb RNAsince the sequence possessed a polyA addition signal just prior to thepoly A tract. The 5′RACE did not revealed any band. The ClontechMarathon cDNA amplification kit was used to prepare a cDNA library from1 hr. adherent macrophages. PCR used a 1040 bp PCR fragment isolatedwith sense and antisense oligonucleotide primers from the determinedcDNA sequence (5′AGCAGGAGCCTTCTCAGGAG 3′ (SEQ ID NO: 13) and5′GATCCAGGGAGGAGCTTGTCC 3′ (SEQ ID NO: 14)) and the universal primerfrom the kit. This resulted in the isolation of a fragment 60 bp longeron the 5′ end than the original 1040 bp fragment.

Human TRELL Cloning

A search of the EST data base showed 1 human clone that was clearlyhomologous to the murine sequence. The clone 154742 (Genbank accessionno: R55379) has a 345 bp sequence 89% homologous to the murine cDNA. Twoprimers derived from the EST (5′CCCTGCGCTGCCTGGAGGAA 3′ (SEQ ID NO: 15)and 5′AGACCAGGGCCCCTCAGTGA 3′ (SEQ ID NO: 16)) were used to screen byRT-PCR different tissues and libraries for the presence of hTRELLtranscripts. Correct size products were obtained from liver, spleen,lymph node, THP-1 and tonsil, but not from U937 mRNA. The 201 bp productwas cloned and used to screen a lambda gtl10 human tonsil cDNA library.10⁶ plaque forming units were plated at 10⁵ PFU/plate. Duplicate liftswere made onto 20×20 cm nitrocellulose filters and hybridized with aprobe prepared by random-priming. The filters were hybridized overnightat 65/C in plaque screen buffer (50 mM Tris pH 7.5, 1 M NaCl, 0.1%sodium pyrophosphate, 0.2% polyvinylpyrolydone and 0.2% Ficoll)containing 10% dextran sulphate, 100 mg/ml tRNA and 6×10⁵ cpm/ml ofprobe. They were washed twice with plaque screen buffer and twice with2×SSC, 0.1% SDS at 65/C. Lambda miniprep DNAs were prepared frompositive colonies and the clones with the largest inserts were selectedfor large scale DNA purification and DNA sequencing. The inserts weresubcloned into the Not1 site of pBlueScript SK+. One human EST (R55379)was found encoding parts of the human TRELL sequence.

RNA Analysis

Either a 0.45 kb PpuM1/BstX1 or a 1.25 Narl/NotI fragment of the hTRELLcDNA was labeled by random priming and used to probe human and mousetissue Northern blots purchased from Clontech. Mouse tissues and cellswere RNA-extracted with TRI-reagent. Northern analysis were doneessentially as already described (Chicheportiche and Vassalli, 1994)with 4 μg of total RNA and ³²P labeled random primed mTRELL cDNA.

Chromosomal Assignment

A panel of DNA from monochromosomal cell hybrids (HGMP Resource centre,Hinxton, Cambridge, UK) was used to amplify by PCR a 340 bp fragmentwith primers chosen in 3′ untranslated region that are not homologous tothe murine sequence (5′AGTCGTCCCAGGCTGCCGGCT 3′ (SEQ ID NO: 17) and5′CCTGAAGTGGGGTCTTCTGGA 3′ (SEQ ID NO: 18)). Amplification was done for40 cycles, 30 sec at 94/C, 90 sec at 65/C and 90 sec at 72/C. Detectionwas carried out on ethidium bromide stained agarose gel.

Expression of Recombinant hTRELL Protein

A soluble expression construct combining the VCAM leader sequence, themyc peptide tag and the extracellular domain of hTRELL similar to thatdescribed for lymphotoxin-b (ref) was prepared in a manner similar tothat described for LTh (Browning et al., 1996). The following DNAfragments were isolated, a Not1/blunt fragment encoding the VCAM leaderand a pair of oligonucleotides encoding the myc tag (5′ blunt, 3′ PpuM1site) which have been described, a 0.45 kb PpuM1/BstX1 fragment of TRELLand a 0.65 BstX1/Not1 fragment of TRELL. The four fragments were ligatedinto a Not1/phosphatased pBluescript vector. The Not1 insert from thisvector was transferred into the pFastBac™ 1 vector (GibcoBRL) and usedto generate recombinant baculovirus. Soluble TRELL was prepared byinfecting HiFive™ insect cells at a MOI of 10 and the medium washarvested after 2 days. The following items were added to the media:HEPES buffer to a final concentration of 25 mM, pH 7.4, 1 mM AEBSF(Pierce) and 1 mg/ml pepstatin. The media was filtered and concentratedten fold by ultrafiltration over a Amicon 10 kDa cutoff filter.Concentrated TRELL containing medium was directly loaded onto a SPsepharose Fast Flow column and washed with 25 mM HEPES buffer pH 7.0containing 0.4 M NaCl. TRELL was eluted with the same buffer with 0.6 MNaCl. Purified TRELL was subjected to sizing analysis.

Analysis of Secretion

Vectors for EBNA based expression were constructed using the vectorCH269 which is a modified version of the pEBVHis ABC (Invitrogen)wherein the EBNA gene and the histidine tag were removed. A 0.71 kbfragment of hTNF in the pFastBac vector was provided by Dr. P.Pescamento and A. Goldfeld. The SnaBI/XhoI insert was ligated into thePvuII/XhoI site of CH269. A genomic TNF insert containing the 1-12cleavage site deletion was a gift from Dr. G. Kollias and was insertedinto the CH269 vector by A. Goldfeld. A 1.8 kb Not1 insert of hTRELLclone A2A, the 0.98 kb NotI fragment containing the hCD40L cDNA providedby Dr. E. Garber and a 1.46 kb NotI insert containing hLTa (Browning etal., 1995) were ligated into the NotI site of CH269. A 0.81 kb HindIIIinsert containing the hLTh coding region with a modified start site(Browning et al., 1995) was ligated into the HindIII site of CH269.EBNA-293 cells were transfected with the various CH269 vectors alongwith the GFP vector using lipofectamine and either removed with PBS with5 mM EDTA for FACS analysis or after 2 days the cells were subjected tometabolic labelling. Both procedures utilized the following antibodies,hTRELL a rabbit polyclonal Ig fraction, hTNF the mAb 104c, hLTa the mAbAG9, LTa1/b2 the mAb B9 and CD40L the mAb 5C8. FACS analysis was carriedout in RPMI medium containing 10% FBS and 50 μg/ml heat aggregated humanIgG with the antibodies at 5 μg/ml. Phycoerythrin labelled anti-mouse orrabbit IgG (Jackson ImmunoResearch) was used to detect antibody binding.GFP bright transfected cells were live gated. For immunoprecipitation,cells 2 days after transfection were washed with PBS and transferredinto met/cys free MEM containing 200 uCi/ml TranSlabel (ICN). Thesupernatants were harvested and subjected to immunoprecipitation asdescribed (Browning et al., 1995).

Cytotoxicity Assays:

Cell growth assays were carried out as previously described (Browningand Ribolini, 1989). For microscopy, HT29-14 cells were seeded into 12well plates at a density of 200,000 cells/well and grown for 2 days.Human TRELL, TNF, lymphotoxin-a1b2 (Browning et al., 1996) or anti-fas(CH11, Kamaya) were added along with 80 units/ml of human interferon-g.After 26 h, the medium was removed which after cytokine or anti-fastreatment included many dead cells that had detached from the plastic.The remaining cells were fixed with 80% ethanol and washed into PBScontaining 1 mg/ml Hoescht dye. After 2 min the dye was removed, cellswere washed into PBS and examined by fluorescence microscopy.

TABLE II Human TRELL Binding Sites and Cytotoxic Effects on Various CellLines Cell Line Type TRELL Binding Cytotoxicity^(a) Hematopoietic JurkatT lymphoma − − SKW 6.4 EBV B cell − Namalwa Burkitt lymphoma − − K562promyelocytic + − THP-1 monocytic leukemia ++ − Nonhematopoietic HT29colon adenocarcinoma − −++^(b) ME-180 cervical carcinoma − Hela cervicalcarcinoma −^(d) MCF-7 breast adenocarcinoma +/− 293 embryonic kidneycells + nd^(c) Cos kidney fibroblasts + nd^(c) “−” = nobinding/cytotoxicity; “+” = some binding/cytotoxicity; and “++” =significant binding/cytotoxicity ^(a)3-5 day proliferation assay in thepresence and absence of human interferon-g. ^(b)Cytotoxicity was onlyobserved in the presence of interferon-g. ^(c)ND, not determined.^(d)Morphology changes

TABLE III Grouping of Various TNF Family Members by CytotoxicityPatterns Group Receptor Activation Potent inducers of apoptosis TNF,Fas, TRAIL-R^(a), DR-3 in many cell types Weak inducers LTb-R,TRELL-R^(a), CD30 only in limited cell types Cannot induce cell death,CD27, CD40, OX-40 anti-proliferative in some settings ^(a)Thesereceptors have not yet been identified.

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1. A substantially pure polypeptide comprising an amino acid sequencethat is at least 90% identical to amino acids 139 to 284 of SEQ ID NO:4,wherein the polypeptide is capable of binding to an HT-29 coloncarcinoma cell and is cytotoxic to said carcinoma cell.
 2. Asubstantially pure polypeptide comprising an amino acid sequence that isat least 95% identical to amino acids 139 to 284 of SEQ ID NO:4, whereinthe polypeptide is capable of binding to an HT-29 colon carcinoma celland is cytotoxic to said carcinoma cell.
 3. The polypeptide of claim 1,wherein the amino acid sequence is at least 90% identical to amino acids81 to 284 of SEQ ID NO:4.
 4. The polypeptide of claim 2, wherein theamino acid sequence is at least 95% identical to amino acids 81 to 284of SEQ ID NO:4.
 5. The polypeptide of claim 2, wherein the amino acidsequence is amino acids 81 to 284 of SEQ ID NO:4 with a single aminoacid substitution, deletion or insertion.
 6. The polypeptide of claim 2,wherein the amino acid sequence is amino acids 81 to 284 of SEQ ID NO:4with one conservative substitution of an amino acid.
 7. The polypeptideof claim 1, wherein the amino acid sequence is at least 90% identical toamino acids 36 to 284 of SEQ ID NO:4.
 8. The polypeptide of claim 2,wherein the amino acid sequence is at least 95% identical to amino acids36 to 284 of SEQ ID NO:4.
 9. A substantially pure polypeptide thatcomprises amino acids 80 to 225 of SEQ ID NO:2.
 10. The polypeptide ofclaim 9, wherein the polypeptide comprises amino acids 22 to 225 of SEQID NO:2.
 11. The polypeptide of claim 10, wherein the polypeptideconsists essentially of amino acids 22 to 225 of SEQ ID NO:2.
 12. Thepolypeptide of claim 11, wherein the polypeptide consists of amino acids22 to 225 of SEQ ID NO:2.
 13. A substantially pure polypeptidecomprising an amino acid sequence at least 90% identical to amino acids80 to 225 of SEQ ID NO:2, and the polypeptide is capable of binding toan HT-29 colon carcinoma cell and is cytotoxic to said carcinoma cell.14. The polypeptide of claim 13, wherein the polypeptide comprises anamino acid sequence that is at least 90% identical to amino acids 22 to225 of SEQ ID NO:2.
 15. A substantially pure polypeptide comprising anamino acid sequence at least 95% identical to amino acids 80 to 225 ofSEQ ID NO:2, wherein the polypeptide is capable of binding to an HT-29colon carcinoma cell and is cytotoxic to said carcinoma cell.
 16. Thepolypeptide of claim 15, wherein the polypeptide comprises an amino acidsequence at least 95% identical to amino acids 22 to 225 of SEQ ID NO:2.17. The polypeptide of claim 2 or claim 8, wherein the polypeptidefurther comprises an amino acid tag sequence.
 18. The polypeptide ofclaim 2 or claim 8, wherein the polypeptide comprises a type I or typeII leader sequence.
 19. A composition comprising the polypeptideaccording to claim 2 or claim 8, and a pharmaceutically acceptablecarrier.
 20. The composition of claim 19, wherein the composition isformulated for injection.
 21. The composition of claim 19, wherein thecomposition contains a physiologically compatible buffer.
 22. Asubstantially pure polypeptide comprising an amino acid sequence that isat least 90% identical to amino acids 139 to 284 of SEQ ID NO:4.
 23. Thepolypeptide of claim 22, wherein the amino acid sequence is at least 90%identical to amino acids 81 to 284 of SEQ ID NO:4.
 24. The polypeptideof claim 22, wherein the amino acid sequence is at least 90% identicalto amino acids 36 to 284 of SEQ ID NO:4.
 25. A substantially purepolypeptide comprising an amino acid sequence at least 90% identical toamino acids 80 to 225 of SEQ ID NO:2.
 26. The polypeptide of claim 25,wherein the polypeptide comprises an amino acid sequence at least 90%identical to amino acids 80 to 225 of SEQ ID NO:2.
 27. The polypeptideof claim 25, wherein the polypeptide comprises an amino acid sequence atleast 90% identical to amino acids 22 to 225 of SEQ ID NO:2.